1. Field of the Invention
This invention relates to novel polynucleotide constructs, isolated polynucleotides, plant cells containing said polynucleotides, and plants derived therefrom, wherein the polynucleotide constructs comprise a sequence of a hemicellulase, .beta.-(1,4)-endoglucanase, gene from pepper (Capsicum annuum). The invention also provides a method of altering a phenotype of a plant by modulating expression of an endogenous hemicellulase gene. In particular it involves the use of recombinant DNA technology to control hemicellulase expression in plants. The invention also provides a method for producing compositions of pepper hemicellulase, cells capable of expressing pepper hemicellulase, and compositions thereof.
2. Description of the Prior Art
Plant development is a complex physiological and biochemical process which involves the coordinated expression of many genes. Ripening, the final phase of fruit development, involves a number of metabolic changes in fruit tissue. These metabolic changes can result from alterations in expression of various genes; the alterations can involve transcriptional and/or post-transcriptional control (e.g., RNA stability, translation, etc.).
An important aspect of the ripening process is fruit softening, which occurs in conjunction with modifications of the cell wall of cells in the fruiting body. Many subtle changes in metabolic activity are involved in this response, and the process of ripening and its control remain incompletely characterized.
For many years, scientists have sought to understand better the process of ripening and to eventually develop methods and plant varieties which were amenable to manipulation of the ripening process. Control of fruit ripening would permit industry to employ more convenient handling, storage, and shipping methods, and ensure that consumers were provided with fruit of suitable ripeness while minimizing wastage resulting from damaged or overripe fruit.
Various types of fruiting plants have been investigated to delineate the genes which control ripening in each plant variety. Several enzymes have been implicated in the ripening process in some plants, however different genes or sets of genes may be causally involved in ripening in different plant varieties.
The prior art discloses ripening-impaired mutants, such as the rin mutant which have been used to study fruit ripening. Tigchelaar Hortic. Sci. 13: 508 (1978). The use of these mutants to specifically control fruit softening has met with limited success, however, because of the pleiotropic nature of these mutations.
Methods for controlling the expression of certain plant genes can be used to modify a plant's phenotype as desired, such as controlling the rate or time at which fruit ripening occurs. One way to control expression of endogenous plant genes is the inhibition of specific gene expression by antisense suppression (U.S. Pat. Nos. 5,457,281, 5,453,566, 5,365,015, 5,254,800, 5,107,065, and 5,073,676). An alternative method to inhibit expression of specific genes is sense suppression (U.S. Pat. Nos. 5,283,184, 5,231,020, and 5,034,323).
An increase in the activity of polygalacturonase, an enzyme responsible for the degradation of pectin, has been correlated with fruit softening. Recombinant constructs have been prepared containing a plant promoter linked to polygalacturonase cDNA in the antisense direction. These constructs have been inserted into tomato to inhibit the activity of this enzyme in ripening fruit. (Smith et al. (1988) Nature 334: 724; Sheehy et al. (1988) Proc. Nat. Acad. Sci. 85: 8805; Hiatt et al., U.S. Pat. Nos. 4,801,340, 5,387,757, and 5,457,281; Bridges et al., EPO Publication No. 271,988. U.S. Pat. No. 5,304,490 discusses DNA constructs which contain various genes believed to be involved in the process of fruit ripening. U.S. Pat. No. 5,438,152 discloses methods and compositions for controlling pigment, blossom and scar size, and disease resistance in plants.